Our long-term objectives on this project are to explore the molecular biology of membranes, to provide molecular mechanisms for the processes of active transport through membranes, the mechanochemistry of membranes, and cell activation of hormones, mitogens, and other ligands. We propose to carry out the following specific studies: 1) to investigate a molecular mechanism of active transport, the "aggregate-rearrangement" mechanism, using the Ca-ATPase transport protein of sarcoplasmic reticulum membranes. Fluorescence probes will be covalently attached to the protein both in intact membranes and in the isolated state. The latter will be reconstituted in synthetic lipid vesicles. In both cases, the probes will be used to monitor the postulated rearrangements occurring in transport. 2. to study the localization of a new smooth-muscle protein, filamin, in non-muscle cells, and its role in mechanochemical processes of these cells; 3) to develop methods of improving ultrastructural cytochemistry through the use of ultrathin sections prepared by ultracryotomy. BIBLIOGRAPHIC REFERENCES: A disulfide-brige bifunctional imidoester as a reversible cross-linking reagent, Arnold Ruoho, Paul A. Bartlett, Anne Dutton, and S.J. Singer, Biochem. Biophys. Res. Commun. 63:417 (1975). Detection and ultrastructural localization of human smooth muscle myosin-like molecules in human non-muscle cells by specific antibodies, Richard G. Painter, Michael Sheetz and S.J. Singer, Proc. Nat. Acad. Sci. 72:1359 (1975).